IRES sequences with high translational efficiency and expression vectors containing the sequence

ABSTRACT

An isolated and cloned translation control element, and analogues thereof, having the nucleotide sequence as set forth in SEQ ID NO:7 and designated SP163, are disclosed. The translation control element controls cap-independent mRNA translation via an internal ribosome entry site (IRES). The present invention provides expression vectors comprising the translation control element SP163 or its analogues operatively linked to a gene sequence to be expressed. In alternative embodiments, the expression vector comprises at least two nucleic acid sequences to be translated and SP163 is operatively linked to at least one of the sequences to be translated. The sequences to be translated may be linked to only one promoter in an embodiment. The present invention further provides a method for facilitating and enhancing cap-independent translation of mRNA by including in an expression cassette a translation control element having the nucleotide sequence as set forth in SEQ ID NO:7 and designated SP163.

This application is a §371 national stage of PCT/US98/03699 filed Feb.25, 1998, which claims the benefit of U.S. Provisional Application No.60/038,500, filed Feb. 25, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an isolated and cloned DNA sequenceelement that can be incorporated into expression vectors for the purposeof improving translation of a given mRNA and to enable the translationof the mRNA in a cap-independent manner.

2. Description of Related Art

There is an extensive and growing need to produce eukaryotic geneproducts in eukaryotic cells. Methods are needed to maximize expressionof a desired gene and production of the gene product in eukaryoticcells. Initiation of eukaryotic protein synthesis involves about teninitiation factors, eIFs. The eIF4 group of initiation factorscollectively catalyze the recognition of the mRNA cap, the unwinding ofmRNA secondary structure, and the binding of mRNA to the 43Spreinitiation complex [Pain, 1996]. The selection of a particular mRNAfrom the pool of translatable RNAs is determined by the relativeefficiency of translation initiation by ribosome scanning and is largelygoverned by the composition and structure of the 5′-untranslated region(5′UTR) of the mRNA [Kozak, 1991; Sonenberg, 1996].

In certain instances translation of the transcribed, processed, andend-modified (i.e. capped and polyadenylated) mRNA is the limiting stepin the production of the protein [Mathews et al., 1996; Meyuhas et al.,1996]. Translation is initiated by mRNA-protein interactions precedingthe engagement of the small ribosomal subunit (40S) with the mRNA. Thus,limited availability of cap-binding proteins and competition with othercellular mRNAs for that proteins can be a rate-limiting factor intranslation of the desired protein. This limitation can be even morepronounced under stress conditions (e.g. heat shock, hypoxia, nutrientdeprivation) in which cap-dependent translation is markedly compromised.[Mathews et al., 1996; Meyuhas et al., 1996].

An alternative mode of translation is one in which ribosomes bind to themRNA independent of the cap structure using an internal ribosome entrysite (IRES), a specialized sequence within the 5′ untranslated regionsthat directly promote ribosome binding, independent of a cap structure.IRES elements were first discovered in picornaviral mRNAs which atenaturally uncapped but nonetheless efficiently translated [Jang et al.,1988; Pelletier and Sonenberg, 1988; Oh and Sarnow, 1995]. Subsequently,it was found that some cellular RNAs which are normally capped can betranslated either by the 5′ end-dependent scanning mechanism or by aninternal ribosome binding mechanism. Generally, IRES cannot beidentified by sequence homology; known IRES have been identified anddefined functionally [Mountford and Smith, 1995]. It appears that it isthe conformation of the IRES sequence that enables the binding on theribosome.

The list of cellular genes shown to contain sequences mediating internalinitiation within their 5′UTR includes the immunoglubulin heavy chainbinding protein (BiP) [Macjak and Sarnow, 1991], anntennapedia [OH etal., 1992], fibroblast growth factor (FGF) [Vagner et al., 1995],platelet-derived growth factor-B (PDGF-B) [Bernstein et al., 1997],insulin-like growth factor II (IGF-II) [Teerink et al., 1995], and thetranslation initiation factor eIF4G [Gan and Rhoads, 1996]. Thepotential utility of a cap-independent translation mode is bestdemonstrated in the case of viral RNAs in circumstances wherecap-dependent translation is completely abrogated (through cleavage ofan essential cap-binding protein by a virus-encoded protease) and thetranslation machinery is taken-over by IRES-containing viral RNA[Pelletier and Sonenberg, 1988]. The option of internal initiation is anadvantage for competition with other mRNAs when certain components ofthe eIF4 complex become rate-limiting and this option provides a givenmRNA the ability to be translated at times when cap-dependenttranslation is compromised. Such circumstances may develop under hypoxiawhere overall protein synthesis is significantly inhibited [Heacock andSutherland, 1988; Kraggerud et al., 1995] or other stress conditions.

It would be useful to have a small sequence element, derived from anaturally-occurring cellular 5′UTR, that endows any desired gene withthe ability to be more efficiently translated, in general, and to betranslated in a cap-independent manner, in particular. That is, it wouldbe useful to have additional IRES sequences with high translationalefficiency, to use in expression vectors, to control mRNA translationand therefore protein synthesis as well as in gene therapy vectors.

SUMMARY OF THE INVENTION

According to the present invention, an isolated and cloned translationcontrol element, and analogues thereof, having the nucleotide sequenceas set forth in SEQ ID No:7 and designated SP163, is disclosed. Thetranslation control element controls cap-independent mRNA translationvia an internal ribosome entry site (IRES). The present inventionprovides expression vectors comprising the translation control elementSP163 or its analogues operatively linked to a gene sequence to beexpressed. In alternative embodiments, the expression vector comprisesat least two nucleic acid sequences to be translated and SP163 isoperatively linked to at least one of the sequences to be translated.The sequences to be translated may be linked to only one promoter in anembodiment.

The present invention provides a method for facilitating and enhancingcap-independent translation of mRNA by including in an expressioncassette a translation control element having the nucleotide sequence asset forth in SEQ ID No:7 and designated SP163.

The novel sequence element (designated SP163) is composed of sequencesderived from the 5′-UTR of VEGF (Vascular Endothelial Growth Factorgene), however, in a novel arrangement that was presumably generatedthrough a previously unknown mode of alternative splicing. Functionalanalysis has shown that SP163 functions as a significant stimulator oftranslation and as a mediator of cap-independent translation.Imterestingly, the full-length 5′-UTR of VEGF has fair IRES activityindicating that these stronger activities were generated by a specificmolecular event causing the juxtaposition of two specific 5′-UTRsegments.

The advantages of SP163 is that it is a natural cellular IRES elementwith a superior performance as a translation stimulator and as amediator of cap-independent translation relative to known cellular IRESelements. Another advantage of SP163 is that these functions aremaintained under stress conditions.

DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

FIGS. 1A-1B are schematic diagrams of vectors and constructs used inthis study. FIG. 1A is bicistronic expression vector in which expressionof a bicistronic mRNA is driven by a CMV virus promoter. A fireflyluciferase (LUC) is translated from the first cistron and a secretedalkaline phosphatase (SeAP) from the second cistron. Putative IRESelements are inserted into the intercistronic space (ICS). FIG. 1B isMonocistronic expression vector in which SeAP expression is driven by aCMV virus promoter. Sequence elements tested for atranslation-modulating activity are inserted upstream of the SeAP codingregion.

FIGS. 2A-2C are graphs showing the production of Luciferase and SeAPfrom a bicistronic mRNA in stably transfected C6 cells wherein FIG. 2Ais a graph of production of SeAP from the downstream cistron. Pools ofstably transfected C6 clones were grown to 70% confluence, medium wasreplaced with a fresh medium (t=0) and aliquots were withdrawn at theindicated time points and analyzed for SeAP activity. Activity isexpressed as cumulative units of SeAP in 1 ml of medium and isstandardized to total protein. For testing SeAP production underhypoxia, cells were shifted to 1% oxygen at t=0 and further analyzed asabove. FIG. 2B is a bar graph showing ratio of SeAP/LUC production inthe different transfectants. SeAP/LUC ratio was calculated from therespective activities determined at the endpoint of the experiment (t=24hrs.). FIG. 2C is a bar graph showing that under hypoxia stress wheretranslation of LUC gene from the upstream cistron was reduced the rateof SeAP production was unaffected indicating internal initiation.Plasmid designation is as shown in FIG. 1. N=Normoxia, H=Hypoxia.

FIGS. 3A-3B are photographs of Northern blot analysis of mRNAstranscribed from bicistronic plasmids. RNA was extracted fromuntransfected C6 cells and from the same stably transfected C6 poolsused in the experiment shown in FIG. 2. 20 μg of each RNA waselectrophoresed in two parallel lanes, blotted and hybridized witheither a LUC-specific probe (A) or a SeAP-specific probe (B). To assureequal loading, ribosomal RNAs were stained with methylene blue prior tohybridization. Lane 1—untransfected C6 cells; Lane 2—B/0; Lane 3—B/UTR;Lane 4—B/Bip.

FIG. 4 is a photograph of a Northern blot analysis of VEGF mRNA speciesexpressed under conditions of severe hypoxia. C6 cells were grown undernormoxia (N) or were exposed to 1% oxygen for 4 hours (4 H) or 16 hours(16 H). 5 μg of poly (A)⁺ RNA from each culture was subjected to aNorthern blot analysis using a VEGF-specific probe. The bold arrowpoints at the most frequently encountered 3.7 kB VEGF mRNA.

FIG. 5 is a graph of IRES activity of SP163. 293 cells were transientlytransfected with each of the indicated bicistronic plasmids and SeAP andLUC activities were determined 36 h post-transfection as described under‘Methods’. Results are expressed as a SeAP/LUC ratio and represent theaverage of 4 independent transfections for each plasmid. Plasmiddesignations are as shown in FIG. 1. B/ΔSP163 is a deletion mutant ofSP163 missing the first 31 nucleotides. B/SP163/M5′ is a substitutionmutation wherein the 9 nucleotides from the 5′ teminus of SP163 aresubstituted. B/SP163/M3′ is a substitution mutation of the 5 nucleotidesfrom the 3′ terminus. Inset is a photograph of a Northern blot analysiswith a SeAP-specific probe and a LUC-specific probe performed on 20 μgof the total RNA extracted from transfected 293 cells 36 hpost-transfection. Lane 1—mock-transfected cells; lane 2—B/0-transfectedcells; lane 3—B/UTR-transfected cells; lane 4—B/SP163-transfected cells.

FIG. 6 is a graph showing the effectiveness of SP163 when compared toanother cellular IRES, that of the grp78 gene.

FIG. 7 is a graph showing results obtained for two representativespecies, rat C6 cells and human Hela cells.

FIGS. 8A-8B are graphs showing that the enhancing performance of SP163is retained when the expression vector is stabely integrated into thehost cell genome.

FIG. 9 is a graph showing the relative stimulation of SeAP activity andSeAP activity in pBKC/163S was 4 fold higher than pCIBB/S and over6-fold higher than pCImc/S.

FIGS. 10A-10C are photographs of gel electrophoresis showing Northernblot analysis of the amounts of mRNA of the reporter genes (SeAP andLuc) that are transcribed from the different vectors.

FIG. 11 is a graph showing translation enhancing activity of VEGF 5′UTRsequences under normal (N) and hypoxia (H) conditions. C6 cells werestably transfected with the monocistronic constructs indicated. SeAPactivity was analyzed in pools of transfected clones as described under‘Methods’ and values were standardized to total protein. For analysis ofSeAP activity under hypoxia, cells were shifted to 1% oxygen 24 hoursbefore sampling. Inset is a photograph of Northern blot analysis ofSeAP-containing mRNAs in stably-transfected pools. Lane1—M/0-transfected cells; lane 2—M/UTR-transfected cells; lane3—M/SP163-transfected cells; lane 4—non-transfected cells.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, an isolated and cloned translationcontrol element, and analogues thereof, having the nucleotide sequenceas set forth in SEQ ID No:7 and designated SP163, is disclosed. Thetranslation control element controls cap-independent mRNA translationvia an internal ribosome entry site (IRES).

The term Analogue as used herein is defined as a nucleotide sequencevariant (alternatively the terms alteration, sequence alteration,sequence variant can be used) with some differences in their nucleotidesequences as compared to the native sequence of SEQ ID No:7 but, suchthat the conformational folding of the sequence, to allow its use as anIRES, is not compromised. Ordinarily, the analogue will be generally atleast 70% homologous over any portion that is functionally relevant. Inmore preferred embodiments the homology will be at least 80% and canapproach 95% homology to the nucleotide sequence. The nucleotidesequence of an analogue may differ from that of the translation controlelement of the present invention when at least one nucleotide isdeleted, inserted or substituted, but the translation sequence elementremains functional as an IRES. Functionally relevant refers to thebiological property of the sequence and in this context means aconformational or other aspects of the molecule that allow functioningas an enhanced IRES as described herein.

The present invention provides expression vectors comprising thetranslation control element, designated SP163, operatively linked to agene sequence to be expressed. In alternative embodiments the expressionvector comprises at least two nucleic acid sequences to be translatedand the translation control element is operatively linked to at leastone of the sequences to be translated. Vectors are known or can beconstructed by those skilled in the art and contain all expressionelements necessary to achieve the desired transcription of the sequencesin addition to the sequence of the present invention as shown in theExamples herein below. The vectors contain elements for use in eitherprocaryotic or eucaryotic host systems depending on their use. One ofordinary skill in the art will know which host systems are compatiblewith a particular vector.

Additional features can be added to the vector to ensure its safetyand/or enhance its therapeutic efficacy as known in the art. Suchfeatures include, for example, markers that can be used to negativelyselect against cells infected with the recombinant virus. An example ofsuch a negative selection marker is the TK gene that confers sensitivityto the anti-viral drug gancyclovir. Negative selection is therefore ameans by which infection can be controlled because it provides induciblesuicide through the addition of antibiotic. Such protection ensures thatif, for example, mutations arise that produce altered forms of the viralvector or sequence, cellular transformation will not occur. Featuresthat limit expression to particular cell types can also be included.Such features include, for example, promoter and regulatory elementsthat are specific for the desired cell type in addition to thetranslation control sequence of the present invention. As shown in theExamples herein the sequences can be used in all cell types in bothvectors for use in cell culture and in vivo gene therapy.

The present invention also provides a method for facilitatingpreferential translation of a gene of interest over the bulk of cellularmRNAs which are not of interest. In other words allowing restriction ofprotein production in the host cell to essentially the protein/peptidecoded for by the gene/nucleotide sequence of interest. The methodprovides the steps of including in an expression vector a translationcontrol sequence of the present invention as set forth in SEQ ID No:7,operatively linked to the gene of interest. The vector is then expressedin host cells as is known in the art. The cells are then treated withcompounds that inhibit cap-dependent translation. The reagents that areadministered to the cell can include, for example, a virus-derivedprotease or anysomicin or other agents.

The present invention provides a second method for facilitatingpreferential translation of a gene of interest over the bulk of cellularmRNAs. In this further embodiment, the steps provide constructing anexpression vector including a translation control sequence of thepresent invention as set forth in SEQ ID No:7, operatively linked to thegene of interest as well as any other required regulatory elementsrequired for the sequence of interest. The vector is then expressed inhost cells, as is known in the art. The host cells are then culturedunder conditions which will induce cellular stress such as hypoxia, heatshock, hypoxia, hypoglycemia, iron deprivation and otherconditions/treatments/compounds which will induce cellular stress suchthat cap-independent mRNA translation is active and cap-dependenttranslation is impaired.

As shown in the Examples, with the aid of reverse transcription-PCR, theuse of mouse cytoplasmic mRNA as a template, and the primers indicatedin Table 1 (corresponding to the 5′ and 3′ boundaries of the5′-untranslated region of VEGF), the sequence of the present inventionshown in Table 1, SEQ ID No:7, was amplified and subsequently cloned.

Comparison of this sequence (designated SP163; SEQ ID No:7) with known5′-UTR VEGF sequences from rat [Levy et al, 1995], mouse [Shima et al,1995] and human [Tischer et al, 1991] revealed that it is composed ofthe first 31 nucleotides of the 5′-UTR (including the cap) adjoined tothe last 132 nucleotides of the 5′UTR (i.e. up to the initiator ATGcondon). It appears that SP163 is generated by a splicing event thatremoves the bulk (>800 nucleotides) of internal 5′-UTR sequences.

Sequencewise, there is no obvious resemblance among the known cellularIRESs, nor do any of the cellular IRESs show a significant homology withpicornavirus IRESs. For example, none of the known cellular IRESs has apyrimidine-rich tract located 25 nucleotides upstream of the initiationsite, as in the picornavirus IRESs [Ehrenfeld, 1996]. VEGF share aconsiderable sequence homology with another cellular gene that has anIRES element namely, PDGF. However, a search for sequence homologybetween the respective 5′UTRs have shown that, despite a similarity inlength and overall high GC content, there is no significant homology inprimary sequences.

Further Examples demonstrate that SP163 significantly improvesproduction of a given protein, when included in various contexts of bothmoncistronic and bicistronic expression vectors. SP163, was introducedinto the basic mono-cistronic expression vector pCIbb, and into the newbi-cistronic expression vectors pBIC-LS to create improved vectors(FIGS. 1A,B; Table 2). Experiments describing the performance of SP163in the two vector systems are summarized as follows and detailed in theExamples.

In the mono-cistronic vector type (pBKC; M/O), FIG. 1B, SP 163 isintroduced between the strong CMV promoter and the target gene. In thisconfiguration, it enhances the expression of the gene locateddownstream. The secreted alkaline phosphatase reporter (SeAP) system isused to measure SP163 activity for testing its performance in differentcellular conditions. This reporter system is especially convenient sinceit monitors the reporter activity in the growth media of the cells andallows the performance of elaborate kinetics studies.

In the bi-cistronic vector system, FIG. 1A, SP163 is introduced betweenthe two target recombinant genes. The expression of the first gene isdriven by the strong CMV promoter, while that of the second is governedby cap-independent translation via the IRES element SP163. In thereporter version, bi-cistronic vector pBIC/LS (B/O; FIG. 1A), SP163 orany other IRES fragments studied are cloned into the uniquemultiple-cloning site element downstream to the luciferase (Luc) gene.The IRES element directs the expression of Secreted Alkaline Phosphatase(SeAP) via a cap-independent mechanism. The activity of the luciferasegene is used to normalize changes in transcription between differentvectors containing different IRESs.

This embodiment of pBIC with the IRES of the present invention willallow the cloning of any two target genes, such as recombinant genes Iand II, so that gene I will be expressed via the CMV promoter and geneII will be expressed via SP163.

Transient transfection is commonly used for characterization of thefunction of a cloned gene. In order to analyze the ability of SP163 tostimulate expression in transient transfection, the vectors pBKCS andpBKC163S were introduced into different cell lines by liposomes mediatedtransfection. As shown in the Examples, the results obtained indicatethat in all the species tested the addition of SP163 to the pBKC/Svector leads to a significant stimulation of expression of 5-10 fold asmeasured by the reporter SeAP.

In the case of VEGF, internal ribosome entry circumvents the need for atroublesome ribosome scanning through the exceptionally long, highlystructured 5′UTR which could have rendered translation an inefficientprocess [Kozak, 1991]. Among its many functions, VEGF plays an importantrole in maintaining vascular homeostasis and controlling vascularpermeability [Senger et al., 1993]. Notably, its unscheduleddownregulation at critical developmental timings may cause pathologicregression of existing vessels [Alon et al., 1995]. Thus, the capacityfor cap-independent translation may serve as a safeguard, preventing thedeleterious consequence of VEGF under-translation in circumstances wherecap-dependent translation is transiently compromised.

The above discussion provides a factual basis for the use of IRESsequences with high translational efficiency and expression vectorscontaining the sequence. The methods used with and the utility of thepresent invention can be shown by the following non-limiting examplesand accompaning figures.

EXAMPLES

General Methods

General methods in molecular biology: Standard molecular biologytechniques known in the art and not specifically described are generallyfollowed as in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992), and in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989). Polymerase chain reaction (PCR) is carried outgenerally as in PCR Protocols: A Guide To Methods And Applications,Academic Press, San Diego, Calif. (1990). Reactions and manipulationsinvolving other nucleic acid techniques, unless stated otherwise, areperformed as generally described in Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, andmethodology as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202;4,801,531; 5,192,659 and 5,272,057 and incorporated herein by reference.In-situ (In-cell) PCR in combination with Flow Cytometry can be used fordetection of cells containing specific DNA and mRNA sequences [Testoniet al, 1996, Blood 87:3822]. The vectors of the present invention aresynthesized as described herein and by any method known in the art.

Construction of bicistronic and monocistronic expression plasmids. Thebasic bicistronic vector (designated B/0) and basic monocistronic vector(designated M/0) used in this study for insertion of 5′UTR elements wereprovided by QBI Enterprises Inc. (Rehovot, Israel). Their structures aredescribed in FIGS. 1A&B.

5′UTR elements were obtained by RT-PCR amplification using the specificprimers indicated below. Primers were designed in a way that eachamplified fragment was bounded by XhoI (5′) and NcoI (3′) sites and wasinserted between the XhoI (5′) and BsmBI (3′) sites of theintercistronic spacer of the bicistronic vector B/0 or into the samesites of the monocistronic vector M/0. In either case, the hybridNcoI/BsmBI site recreated the initiator ATG codon of the SeAP cistron.

Cloning VEGF and BiP 5′UTRs: RNAs from hypoxic NIH3T3 cells or 293cells, respectively, were reverse transcribed using 10 μg of total RNA.PCR amplification was carried out using a Taq DNA Polymerase possessinga proofreading activity (Pwo DNA Polymerase, BOEHRINGER) and thefollowing oligonucleotide primers: VEGF 5′ primer: 5′CTCGAGCGCAGAGGCTTGGGGC (SEQ ID No:1). VEGF 3′ primer:5′CCATGGTTTCGGAGGCCGTCCG 3′ (SEQ ID No:2) corresponding to nucleotides1218-1235 and 2234-2214, respectively, of the mouse VEGF gene (GeneBankaccession U41383). The oligonucleotide primers used to amplify the fulllength VEGF 5′UTR were also used to amplify the new splice variant ofVEGF 5′UTR designated SP163 which was also inserted into the B/0 and M/0vectors in the same way as described above. BiP 5′ primer:5′CTCGAGAGGTCGACGCCGGCCAAGACA (SEQ ID No:3). BiP 3′ primer:5′CCATGGCTTGCCAGCCAGTTGGGCAGC (SEQ ID No:4) (corresponding tonucleotides 372-392 and 592-572, respectively, of the human BiP gene,Genebank accession M19645).

All plasmid constructs were sequenced to verify their structure and thepreservation of open reading frames within PCR amplified fragments. Asummary of bicistronic and monocistronic constructs used in this studyis shown in Table 2 and FIGS. 1A-B.

Cell culture and DNA transfections: C6 cells, a clonal glial cell linederived from a rat glial tumor [Benda et al., 1968], were grown inDulbeco-Modified Eagle's medium (DMEM) containing 5% fetal calf serumand antibiotics. The human cell line 293 (adenovirus-transformed fetalkidney cells), mouse NIH/3T3 cells and rat primary astrocytes weremaintained in Dulbeco-Modified Eagle's medium (DMEM) containing 10%fetal calf serum and antibiotics. Primary rat astrocytes were preparedas described previously [Frangakis and Kimelberg, 1984].

Where indicated, cells were exposed to hypoxia (1% oxygen) using aCO₂/O₂ incubator (Forma Scientific) in which oxygen levels are monitoredand adjusted automatically.

Transfections were performed using Lipofectin (GIBCO BRL) as follows:Cells were plated at a density of 1-2×10⁵ Cells per 60-mm plate andgrown for 24 hours, medium was replaced with a serum-free DMEM and 200μl of DNA-Lipfctin suspension (containing 2 μg supercoiled plasmid and15 μg of Lipofectin, preincubated at room temperature for 40 minutes)were added in a dropwise manner. Fourteen hours later, medium wasreplaced with a medium supplemented with serum and antibiotics and cellswere incubated for additional 48 hours before splitting into a selectionmedium containing G418 (2mg/ml). Selection continued for 10-14 daysand >50 individual G418-resistant clones were pooled for each plasmid.

For transient transfections, cells were seeded at a density of 2×10⁵cells per 1 mm well and transfected 24 hours later with 1 μg of DNA perwell using the calcium phosphate procedure [Jordan et al., 1996]. Cellsand media were harvested 36 hours post-transfection. Cell extracts wereanalyzed for luciferase activity or used for mRNA preparation (in thelatter case, 10 μg of DNA was used to transfect 2-2.5×10⁶ cells per 100mm plate), while the media was used for analysis of SeAP activity.

RNA analysis: Total RNA was prepared by the guanidiniumthiocyanate/phenol-chloroform extraction procedure [Chirgwin et al.,1979]. mRNA was purified from total cellular RNA using the mRNASeparator Kit (CLONTECH).

Analysis of mRNA in polysomal fractions: Protein synthesis wasinstantaneously arrested by treating the culture with cycloheximide (90μg/ml) for 10 minutes. Cells were collected, washed withphosphate-buffered saline and kept at −70° C. until analyzed. Celllysis, size fractionation of polysomes by sedimentation through sucrosegradients, and preparation of ribosome-associated RNAs were performed asdescribed previously [Meyuhas et al., 1987].

Northern blotting: RNA was denatured in glyoxal and electrophoresedthrough a 1.0% agarose gel. RNAs were transferred onto a nylon-basedmembrane (GeneScreen plus, NEN) by the capillary blot procedure, andwere hybridized with the indicated cDNAs, labeled with ³²P by randomlyprimed DNA synthesis. For standardization, blots were rehybridized witha β-actin probe or, alternatively, ribosomal RNAs were visualized bystaining with methylene blue prior to hybridization. For relativequantification, autoradiograms were scanned using a Phosporlmager (FUJIXBAS 1000 Bio-Imaging Analyzer). Hybridization probes used were: VEGF (a0.6 kB long mouse cDNA fragment encoding VEGF165) [Shweiki et al.,1992], β-actin [Minty et al., 1981], luciferase (a 1.7 kB long cDNAfragment including all of the coding region of firefly luciferase), andSeAP (a 1.5 kB long cDNA fragment including the coding region of thehuman SeAP).

RT-PCR: semi-quantitative RT-PCR was performed using a single tube, onestep RT-PCR system (Access RT-PCR system, PROMEGA) and 20 cycles ofamplification. Oligonucleotide primers used for reverse transcriptionand amplification of the various splicing variants of VEGF mRNA were:

5′GAGAGAATGAGCTTCCTACAG 3′ (SEQ ID No:5) and 5′ TCACCGCCTTGGCTTGTCACA 3′(SEQ ID No:6) (derived from the common fifth and eighth exons,respectively). PCR products were resolved by agarose gelelectrophoresis, and detected by blot-hybridization using aVEGF-specific, ³²P-labeled cDNA probe.

Luciferase and SeAP analysis: Luciferase enzymatic activity in the cellextracts was determined using a commercial Luciferase Assay System(PROMEGA) and the procedure recommended by the supplier. Light generatedwas measured using Lumac/3M BIOCOUNTER M2010-luminometer.

SeAP activity released into the growth medium was determined as follows:medium was heated for 5 minutes at 65° C. and clarified bycentrifugation. Aliquots were diluted in a SeAP buffer (1MDiethanolamine pH 9.8, 0.5 mM MgCl₂, 10m M L-homoarginine) in a 96 wellplate and the enzymatic reaction (at 37° C.) initiated with the additionof 20 μl of 120 mM p-nitrophenylphosphate. The reaction product wasdetermined using an ELISA reader at A450-630. The amounts of conditionedmedium added and incubation times used were adjusted so that readingswere within the linear range of the calibration curve obtained with apurified SeAP standard. Total protein was determined by the Bradfordmethod [Bradford, 1976].

Example 1 Analysis of the Full 5′UTR of VEGF

The 5′UTR of vascular endothelial growth factor (VEGF) has severalfeatures which are incompatible with efficient ribosomal scanning.First, it is considerably longer (1014 nucleotides in the mouse) thanmost eukaryotic 5′UTRs. Second, it has a high G/C content (comprising64% of 5′-UTR sequences and 80% of the 100 nucleotides preceding thetranslation initiation condon) and can potentially form complex stablesecondary structures. Third, the 5′UTR contains a short open readingframe bounded by in-frame initiation and termination condons. Theinherent difficulty for efficient ribosome scanning, on the one hand,and the possibility of IRES, on the other hand, prompted Applicants toexamine if the VEGF mRNA is translated in a cap-independent mode.

In review, VEGF plays a key role in the initiation of blood vesselformation. VEGF is produced by and secreted from the tissue towardswhich new blood vessels eventually extend. A large body of evidencesupports the premise that the amount of VEGF produced determines themagnitude of the angiogenic response through interaction with cognatereceptors expressed on nearby endothelial cells. VEGF expression istightly regulated in vivo and a deviation from the normal levels of VEGFmight be detrimental to the vasculature. For example, a reduction ofVEGF gene dosage by one half (in mice heterozygous for an inactivatingmutation in VEGF) leads to severe vascular defects and early embryoniclethality [Carmeliet et al., 1996; Ferrara et al., 1996]. Conversely,over-expression of VEGF may lead to pathologies like retinopathyresulting from excessive proliferation of blood vessels [Aiello et al.,1994; Pe'er et al., 1995].

VEGF expression can be modulated in vitro by a variety of agents,including a number of cytokines, growth factors and steroid hormones.Yet, from a physiological point of view, regulation of VEGF by hypoxiais the most significant. Inefficient vascular supply and the resultantreduction in tissue oxygen tension, often lead to compensatoryneovascularization acting to satisfy the metabolic needs of the tissue.Hypoxia-induced VEGF was found to be the key mediator of this feedbackresponse [Plate et al., 1992; Shweiki et al., 1992]. VEGF is regulatedby hypoxia at both the transcriptional and post-transcriptional levels.

Transcriptional regulation of VEGF is mediated by the transcriptionfactor, hypoxia-inducible factor 1 (HIF-1) which accumulates underconditions of hypoxia and activates VEGF transcription through bindingto specific promoter sequences [Forsythe et al., 1996]. Hypoxia alsoleads to stabilization of VEGF mRNA [Ikeda et al., 1995; Shima et al.,1995; Stein et al., 1995]. The intrinsically short half-life of VEGFmRNA (approximately 30 minutes) is significantly extended under stress,presumably through hypoxia-augmented binding of yet unidentifiedprotein(s) to its 3′untranslated region [Levy et al., 1996]. Bothmechanisms act to increase steady-state levels of VEGF mRNA. However, itis not known whether production of VEGF is also regulated at the levelmRNA translation and, in particular, whether the mode or extend of VEGFtranslation is effected by hypoxia. VEGF is expected to be maximallyproduced under hypoxia in order to fulfill its function as a mediator ofhypoxia-driven angiogenesis. These considerations led to the examinationof the performance of the 5′UTR under conditions of hypoxic stress.

To determine the efficiency by which VEGF mRNA is translated, primaryastrocyte cultures were grown, cytoplasmic extracts prepared andfractionated by centrifugation through a sucrose gradient intosubpolysomal and polysomal fractions. The relative abundance of VEGFmRNA in these fractions was determined by quantitative RT-PCR. The bulkof VEGF mRNA was associated with polyribosomes, indicating that themajority of VEGF mRNA is engaged in active protein synthesis. Theseresults suggested that VEGF mRNA is efficiently translated under normalgrowth conditions despite its cumbersome 5′UTR.

To determine whether translation of VEGF mRNA is inhibited underconditions of severe hypoxia, a similar analysis was carried out usingcultures grown for 16 hours under 1% oxygen. Astrocytes were chosen forthis study since they are the first cells to respond to hypoxia ofneuronal tissues by upregulating VEGF mRNA expression and are,therefore, the cells responsible for the feedback angiogenic response[Stone et al., 1995]. The fraction of RNA associated with polyribosomeswas reduced by 50% in comparison with cells grown under normoxia. Thisresult is consistent with previous observations that hypoxia causesabout 30-50% inhibition in overall protein synthesis [Heacock andSutherland, 1988; Kraggerud et al., 1995]. Despite the generalimpairment of protein synthesis, the majority of VEGF RNA remainedassociated with polysomes. Notably, all isoforms of VEGF mRNA remainedassociated with the polyribosomal fraction, including the shortestVEGF121 which can accommodate no more than 3-4 ribosomes when translatedat a theoretical maximal rate. In contrast to VEGF, the fraction of mRNAencoding ribosomal protein L19 that was associated with polyribosomeswas reduced 2-fold under hypoxia in comparison to normoxia. Theseresults suggest that translation of VEGF mRNA is not a rate-limitingstep in production of this vital protein. Importantly, translation doesnot appear to be rate-limiting even under hypoxia where the steady-statelevel of VEGF mRNA is elevated by one order of magnitude [Shweiki etal., 1992].

The fact that translation of VEGF mRNA is very efficient despite its‘inappropriate’ 5′UTR, prompted examination of whether VEFG mRNA can betranslated by internal ribosome entry. Bicistronic mRNAs have beeneffectively used in vivo to demonstrate the existence of IRES in bothpicornaviral [Pelletier and Sonenberg, 1988] and cellular [Gan andRhoads, 1996; Macejak and Sarnow, 1991] mRNAs as discussed herein above.A eukaryotic expression vector was constructed in which a CMV viruspromoter drives expression of a bicistronic RNA. The first cistron inthe bicistronic mRNA used in this study, encoded firefly luciferase(LUC) and should be translated by a conventional cap-dependent scanningmechanism. As ribosomes fail to continue scanning through theintercistronic region, the second cistron, encoding a secreted alkalinephosphatase (SeAP), should be translated only if preceded by an IRES.SeAP was chosen as the downstream cistron since secretion of the enzymeinto the culture medium allows accurate quantification and continuousmonitoring of protein synthesis.

To prevent leaky translation into the second cistron, the intercistronicregion included termination condons in all possible reading frames. The5′UTR of VEGF was sub-cloned by precise PCR amplification, inserted intothe intercistronic spacer (ICS) region and transfected into a C6 ratglioma cell line. Pools of stably-transfected clones were prepared, eachcomposed of >50 individual clones. Analysis of pools assured thatpossible differences in gene expression due to different integrationsites are averaged. Reporter gene activity was determined intransfectants obtained with the bicistronic vector alone (designatedB/0) and with the bicistronic vector containing the VEGF 5′UTR(designated B/UTR). The two constructs expressed a bicistronic mRNA fromwhich LUC was translated with a comparable efficiency. Analysis of thedownstream cistron, however, revealed that B/0 produced a negligibleamount SeAP activity, confirming a minimal, if any, readthrough ofribosomes from the LUC to the SeAP cistron. In contrast, B/UTR directedthe production of high levels of SeAP in a continuous manner (FIG. 3A),indicating that the 5′-untranslated region of VEGF contains a functionalIRES element.

To appreciate the relative strength of the VEGF IRES, a comparison wasmade with the well-characterized cellular IRES contained in the 5′UTR ofBiP mRNA [Pelletier and Sonenberg, 1988]. To this end, the 5′UTR of BiPwas inserted into the same site of the bicistronic vector, pools ofstable transfectants were prepared and similarly analyzed for LUC andSeAP production. As shown in FIG. 3B, the VEGF IRES was 5-fold moreefficient than the BiP IRES in directing SeAP production.

While there is a clear rationale for the use of internal initiation inviruses, the advantage of internal initiation in cellular mRNAs is notalways clear. In the case of VEGF, however, the capacity forcap-independent translation might be particularly advantageous inhypoxia where overall protein synthesis is compromised. Therefore, wehave measured SeAP production, presumably by internal initiation, wasunaffected by hypoxia. As shown in FIGS. 2A and 2C, the rate of SeAPproduction, presumably by internal initiation, was unaffected byhypoxia.

To rule out that in B/UTR-transfected cells the protein encoded by thesecond cistron is translated from a monocistronic SeAP mRNA, a RNAblot-hybridization analysis was carried out using both a LUC-specificprobe and a SeAP-specific probe. A single band of RNA was detected ineach case, corresponding to the expected size of the respectivebicistronic transcript (the larger size of hybridized mRNA inB/UTR-transfectants is due to the addition of 1014 base pairs of 5′UTRsequence) (FIGS. 3A,B). Hybridization with both a LUC-specific probe anda SeAP-specific probes detected the same mRNA species, indicating thatboth cistrons are contained in a single transcripts. Importantly, thefailure to detect a band corresponding in size to that of a presumptivemonocistronic seAP mRNA ruled out the possibility that a significantamount of SeAP is translated from a monocistronic SeAP mRNA. Also notethat the inclusion of the 5′UTR had no effect on the steady-state levelof the bicistronic mRNA expressed, indicating that differences inmeasured LUC and SeAP activities are solely due to differences intranslation efficiencies.

Example 2 Identification of SP163

Alternative splicing of VEGF RNA results in formation of four mRNAspecies encoding different isoforms of the protein [Ferrara et al.,1991]. These mRNAs differ with respect to the presence or absence ofcoding exons 6 and 7 and are coordinately regulated by hypoxia (i.e.production of all isoforms is equally increased by hypoxia) [Banai etal., 1994; Minchenko et al., 1994]. There is no evidence, however, forthe existence of variant forms of VEGF mRNA which differ with respect tothe 5′-3′-untranslated regions. Yet, when cells are grown underconditions of severe ischemia, additional new forms of VEGF mRNAaccumulate, the size of which can not be accounted for by changes in thecoding region alone (FIG. 4). Interestingly, smaller VEGF mRNAs havebeen encountered before but have generally been ignored [e.g. Mazure,1996; Finkenzeller, 1995]. The appearance of these smaller mRNAssuggested that they might represent transcripts with a truncated 5′UTR,or a truncated 3′UTR, or both.

To examine the possibility that splice variants of the 5′UTR exist, mRNAfrom hypoxic NIH 3T3 cells was amplified by RT-PCR using oligonucleotideprimers derived from the most distal and most proximal segments of thefull length 5′UTR. The use of this primer combination assured that only5′UTR sequences are amplified and only of transcripts sharing the same5′ terminus. Several fragments were amplified and verified byblot-hybridization as containing VEGF 5′UTR sequences. However, only oneamplified fragment was cloned and further analyzed. The 163 nucleotideslong sequence of this modified 5′UTR, designated SP163 (SEQ ID No:7), isshown in Table 1. Alignment of this sequence with that of the fulllength 5′UTR suggested that it was generated by a splicing event thatjuxtaposed the 5′ cap-containing segment of 31/32 nucleotide next to the132/131 nucleotides preceding the initiator AUG codon. The experimentsdescribed below demonstrate the performance of the SP163 element as amediator of cap-independent translation and its activity as atranslation enhancer.

Example 3 SP163 Function as IRES

As described in Examples 1 and 2, SP163 was identified during the searchfor possible IRES sequences in the 5′UTR of VEGF. A series of furtherexperiments were designed to characterize its activity as an IRES. Theability of SP163 to promote expression that is derived from theribosomal entry site was tested in the bi-cistronic vector pBIC-LS.pBIC-LS contains luciferase gene as the first cistron and SeAP as thesecond cistron, in between is a multiple cloning site that allows theintroduction of potential IRES sequences. The IRES activity of SP163 wascompared to the parental vector pBIC/LS and to the entire 5′UTR of VEGFfrom which it was derived. It has stronger activity than the entire5′UTR. The three plasmid pBIC/LS, pBIC/L5′UTRS and pBIC/L163S wereintroduced transiently into C6 glioma cells. SeAP activity reflects theIRES effectiveness, and luciferase activity is used to normalize in theoverall changes in transcription. SP163 was generally over 5-8 fold moreactive as an IRES than the entire 5′UTR.

The SP163 element was inserted into the ICS region of the samebicistronic vector used to analyze the ‘conventional’ 5′UTR (Example 1).This plasmid, designated B/SP163 was transfected into the human 293 cellline alongside with plasmid B/0 (serving as a negative control) andplasmid B/UTR (for comparison of relative IRES strength). Proteinsencoded by the first and second cistrons were quantified following atransient transfection (FIG. 5). Again, only negligible SeAP activitycould be detected in cells transfected with B/0, confirming a non-leakyribosome scanning through the first cistron. Consistent with the resultsof the stable transfections shown in FIGS. 2A-B, the full-length 5′UTRdirected a considerable level of SeAP production also in cells of humanorigin. Remarkably and unexpectedly, SP163 was 5-fold more efficientthan the full-length 5′UTR as IRES element.

SP163 also functions as an IRES in the CHO cell. pBIC-LS containsneomycin genes that allow selection following introduction andestablishment of the vector into cells. Plasmid pBIC/LS (parental) andpBIC/L163S were transfected into the CHO cell. In this experiment, theeffectiveness of SP163 was compared to another cellular IRES, that ofthe grp78 gene. The result of this experiment, presented in FIG. 6,shows that SP163 function as an IRES after its establishment in the hostgenome. Its activity is over 25-fold over the background (pBIC/LS).Moreover, it is three-fold more effective as an IRES than the IRES ofgrp78, the best characterized cellular IRES known to date.

RNA blot-hybridization analysis was performed to assure thatSP163-directed translation of SeAP was by internal ribosome entry. Asshown in FIG. 5 (inset), a single mRNA species, corresponding in size tothe expected size of a bicistronic B/SP163 mRNA co-hybridized with theLUC-specific probe and the SeAP-specific probe and no monocistronic SeAPmRNA could be detected. This result indicated that SeAP was translatedfrom a bicistronic RNA using a SP163-directed internal ribosome entry.

Example 4 Activity of SP163

Activity in transient expression in cell lines of different species.Transient transfection is commonly used for characterization of thefunction of a cloned gene. In order to analyze the ability of SP163 tostimulate expression in transient transfection, 5 μg of plasmid DNA ofthe vectors pBKCS and pBKC163S was introduced into different cell linesby liposomes mediated transfection. Twenty four hours after thetransfection, the growth media was changed and SeAP activity wasmeasured. Expression was tested in the following cell lines: Rodents—CHO(hamster) and C6 glioma, Primates-Vero, Human Hek 293 and Hela.

The results obtained indicate that in all the species tested, theaddition of SP163 to the pBKC/S vector leads to a significantstimulation of expression of 5-10 fold as measured by the reporter SeAP.In the rat C6 glioma line SP163 addition leads to a 5-fold increase inSeAP activity (FIG. 7), whereas in the Hela Cell line more than 10-foldstimulation was observed (FIG. 7). The conclusion is that SP163 can be auseful addition to any vectors that are used to analyze expression of atarget gene in transient transfection.

Effect of SP163 in stable expression in mono-cistronic vectors.Production of recombinant proteins for commercial purposes is done insystems where expression of the target gene is stable. The parentalexpression vector pBKC/S includes the neomycin gene that confersresistance to G418 (FIG. 1). This feature was used to introduce thepBKC/S and pBKC/163S plasmids into CHO and C6 glioma cell lines. CHO isthe preferred mammalian cell line for the production ofbiopharmaceuticals. Plasmid DNA of the vectors pBKC/S and pBKC163S wasintroduced by the liposome method and colonies resistant to G418 wereselected and analyzed. SeAP expression levels were tested in pools ofthe G418 resistant colonies. The results shown in FIGS. 8A-B and FIG. 11demonstrate that the enhancing performance of SP163 is retained when theexpression vector is integrated into the host cell genome. SP163insertion in the 5′ of the SeAP reporter gene led to a 5-20 foldstimulation of expression in CHO and C6 glioma cells.

The relative stimulation of SeAP activity (FIG. 9), observed in plasmidpBKC/163S is not restricted to the comparison with plasmid pBKC/S. SeAPactivity of pBKC/163S was compared in stable transfection in CHO cellswith an additional two plasmids pCIBB/S to the basic expression vectorand pCImc/S (pCIBB with the multiple cloning site that was engineeredinto pBKC to exclude the possibility that this segment is responsiblefor the enhancing effect). As shown in FIG. 9, SeAP activity inpBKC/163S was four fold higher than pCIBB/S and over six-fold higherthan pCImc/S.

Example 5 SP163 Acts at the Level of Translation in Mono-Cistronic andBi-Cistronic Vectors

SP163 enhances expression by increasing translation efficiency and notby influencing the abundance of mRNA due to increased stability orincreased transcription. This was shown by Northern blot analysis of theamounts of mRNA of the reporter genes (SeAP and Luc) that aretranscribed from the different vectors. The results are shown in FIGS.10A-C.

The results for the mono-cistronic context are presented in FIG. 10A.The amounts of mRNA expressed by plasmids pBKC/S (control) [lane1[,pBKC/5′UTR/S [lane 2], pBKC/163/S [lane 3] and mock transfected cells[lane 4] were the same. The differences in the length of mRNA generatedby the different vectors reflect the addition in length due to theinsert sizes (1 Kb in pBKC/5′UTR/S and 163 bp in pBKC/163/S).

The results for the bi-cistronic vector that are shown in FIGS. 10B and10C indicate that the amounts of mRNA transcribed by the differentcistrons are at the same level. FIG. 10B shows results for theluciferase gene representing the first cistron and panel c for thesecond cistron SeAP (see FIG. 1 for details). Each cistron was analyzedwith the corresponding probe by Northern blot analysis. The amounts ofmRNA were the same for all plasmids: pBIC/LS (control) lane 1,pBIC/LUTRS lane 2, pBIC/L163S lane 3, pBIC/Lgrp78S (lane 5 is mocktransfected cells).

As can be seen from the results in FIGS. 10B and C, only one mRNA isencoded by the bi-cistronic vectors without the IRES DNA (lane 1) orwith the different DNA fragments tested (lane 2, 5′UTR, lane 3 SP163 andlane 4 grp78), clearly indicating that SP163 functions as an IRES andthat the stimulation of expression results from increased translationefficiency of the transcript and not from increase in the amount of thebi-cistronic mRNA or from the generation of a new SeAP mRNA that can betheoretically derived if SP163 has promoter activity.

Example 6 Translational Enhancement by SP163

The possibility that the 5′UTR of VEGF (both the full length form andSP163) act as a translational enhancer was examined by testing itsability to augment translation of a monocistronic mRNA. To this end, thefull-size 5′UTR or SP163 was inserted between a CMV promoter and thecoding region of SeAP in the context of a monocistronic vector (see FIG.1). These constructs, designated M/5′UTR and M/SP163 respectively, weretransfected into C6 cells and analyzed for SeAP production in pools ofstably-transfected clones. Analysis of RNA extracted from transfectedcells detected similar levels of SeAP-containing mRNA which wasrepresented in each case by a single band of the expected size (FIG. 11,inset). In contrast, the amount of SeAP activity released to the culturemedium varied greatly according to the nature of the insert. Thus, thefull-length 5′UTR augmented translation of SeAP by 5-fold and SP163enhanced SeAP translation much greater. In the experimental resultsshown in FIG. 11 enhancement by SP163 was 25-fold and in otherexperiments (not shown) enhancement was up to 40-fold.

Considerations discussed above led to the examination of whether theseelements also function as translational enhancers under conditions ofhypoxic stress. As shown in FIG. 11, enhancement of SeAP translation wasunaffected by growth under conditions of severe hypoxia.

To demonstrate that SP163 is a strong translational enhancer inadditional cell types, the same constructs were also transfected intohuman 293 cells (FIG. 7) and hamster CHO cells (FIG. 8A). SP163 acted asa strong translation enhancer in these cells as well, augmenting SeAPproduction to a level comparable to that shown for C6 cells (data notshown).

Throughout this application, various publications, are referenced bycitation and patents by number. Full citations for the publications arelisted below. The disclosures of these publications and patents in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art to which thisinvention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

TABLE 1 SEQUENCE OF THE SP163 ELEMENT (SEQ ID No:7)

The oligonucleotide primers that were used for amplification of SP163are underlined. These oligonucleotides correspond to the respectiveboundaries of the 1014 nucleotide long mouse VEGF 5′UTR. The initiationATG condon is boxed. Arrows point at two possible locations of apresumptive splice junction. Nucleotide 1 of SP163 corresponds tonucleotides 1218 of the mouse VEGF gene, GeneBank accession U41383,while nucleotide 163 of the SP163 fragment corresponds to nucleotides2231 of the mouse gene.

TABLE 2 BICISTRONIC AND MONOCISTRONIC CONSTRUCTS MonocistronicBicistronic constructs Insert constructs B/0     NONE M/0     B/UTR  

M/UTR   B/SP163

M/SP163 B/BiP   

(ND)

Designations of bicistronic and monocistronic constructs used in theExamples. The nucleotide numbers inside the rectangles represent theinsert length. ND—not done.

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7 1 22 DNA Artificial sequence Artificial sequence (1)..(22) primer 1ctcgagcgca gaggcttggg gc 22 2 22 DNA Artificial sequence Artificialsequence (1)..(22) primer 2 ccatggtttc ggaggccgtc cg 22 3 27 DNAArtificial sequence Artificial sequence (1)..(27) primer 3 ctcgagaggtcgacgccggc caagaca 27 4 27 DNA Artificial sequence Artificial sequence(1)..(27) primer 4 ccatggcttg ccagccagtt gggcagc 27 5 21 DNA Artificialsequence Artificial sequence (1)..(21) primer 5 gagagaatga gcttcctaca g21 6 21 DNA Artificial sequence Artificial sequence (1)..(21) primer 6tcaccgcctt ggcttgtcac a 21 7 166 DNA Artificial sequence Artificialsequence (1)..(166) SP163 7 agcgcagagg cttggggcag ccgagcggca gccaggccccggcccgggcc tcggttccag 60 aagggagagg agcccgccaa ggcgcgcaag agagcgggctgcctcgcagt ccgagccgga 120 gagggagcgc gagccgcgcc ggccccggac ggcctccgaaaccatg 166

What is claimed is:
 1. An isolated and cloned translation control element consisting essentially of the nucleotide sequence as set forth in SEQ ID No:7.
 2. The translation control element as set forth in claim 1 wherein the sequence controls cap-independent mRNA translation via an internal ribosome entry site (IRES).
 3. An expression vector comprising the translation control element as set forth in claim 1 operatively linked to a gene sequence to be expressed.
 4. An expression vector comprising at least two nucleic acid sequences to be translated and said translation control element as set forth in claim 1 and wherein said translation control element is operatively linked to at least one of the sequences to be translated.
 5. The expression vector as set forth in claim 4 wherein a single promoter is included.
 6. A method for facilitating cap-independent translation of mRNA comprising including in an expression cassette a translation control element consisting essentially of the nucleotide sequence as set forth in SEQ ID No:7.
 7. An isolated internal ribosome entry site (IRES) consisting essentially of the nucleotide sequence as set forth in SEQ ID No:7.
 8. A method for facilitating preferential translation of a gene of interest over the bulk of cellular mRNAs by including in an expression vector a translation control element consisting essentially of the nucleotide sequence as set forth in SEQ ID No:7 operatively linked to the gene of interest and expressing the vector in host cells with reagents that inhibit cap-dependent translation.
 9. A method for facilitating preferential translation of a gene of interest over the bulk of cellular mRNAs by including in an expression vector a translation control element consisting essentially of the nucleotide sequence as set forth in SEQ ID No:7 operatively linked to the gene of interest and expressing the vector in host cells under conditions of cellular stress.
 10. An isolated and cloned translation control element consisting essentially of a nucleotide sequence at least 80% homologous to SEQ ID No:7.
 11. The translation control element as set forth in claim 10 wherein the sequence controls cap-independent mRNA translation via an internal ribosome entry site (IRES).
 12. An expression vector comprising a translation control element as set forth in claim 10 operatively linked to a gene sequence to be expressed.
 13. An expression vector comprising at least two nucleic acid sequences to be translated and a translation control element as set forth in claim 10 and wherein said translation control element is operatively linked to at least one of the sequences to be translated.
 14. The expression vector as set forth in claim 13 wherein a single promoter is included.
 15. A method for facilitating cap-independent translation of mRNA comprising including in an expression cassette a translation control element consisting essentially of a nucleotide sequence at least 80% homologous to SEQ ID No:7.
 16. An isolated internal ribosome entry site (IRES) consisting essentially of a nucleotide sequence at least 80% homologous to SEQ ID No:7.
 17. A method for facilitating preferential translation of a gene of interest over the bulk of cellular mRNAs by including in an expression vector a translation control element consisting essentially of a nucleotide sequence at least 80% homologous to SEQ ID No:7 operatively linked to the gene of interest and expressing the vector in host cells with reagents that inhibit cap-dependent translation.
 18. A method for facilitating preferential translation of a gene of interest over the bulk of cellular mRNAs by including in an expression vector a translation control element consisting essentially of a nucleotide sequence at least 80% homologous to SEQ ID No:7 operatively linked to the gene of interest and expressing the vector in host cells under conditions of cellular stress. 